African Journal of Biotechnology Vol. 6 (3), pp. 273-277, 5 Febraury, 2007 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2007 Academic Journals

Full Length Research Paper

Antioxidant and chemoprotective properties of charantia L. (bitter ) extract

Asli SEMIZ* and Alaattin SEN

Department of Biology, Pamukkale University, 20070 Kinikli-Denizli, TURKEY.

Accepted 5 January, 2007

Momordica charantia, commonly known as bitter melon, is used as a vegetable in number of countries. Extracts of M. charantia , fruit pulp, and seed have been reported to have a wide medicinal use in the traditional medical systems, most often as hypoglycemic and anti-diabetic agents. We have studied the effect of M. charantia, collected from Kazdaglari (Mount Ida) in Balikesir, fruit extract on glutathione S-transferases (GSTs), cytochrome P450s (CYPs), and antioxidant enzymes in rats. Male Wistar rats, aged 12 weeks and weighing 200-250 g, were given 200 mg M. charantia fruit extract per kg body weight, i.p., for four consecutive days. At the end of the experimental period, the animals were sacrificed, and liver, kidney, and lung were isolated. Our results have indicated significant increase in especially hepatic antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities. The strongest increase (about 9-fold) was observed in GPx activities while about 2 to 5-fold increases were observed in SOD and CAT. M. charantia fruit extract also exhibited hepatoprotective effects in CCl4-intoxicated rats. In addition, about 50% increase was also noted with hepatic cytosolic GSTs. On the other hand, treatments of rats with M. charantia significantly reduced both ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-deethylase (MROD) activities in rat liver microsomes, which are known to be catalyzed by CYP1A isoforms These results suggest that the M. charantia fruit extract possesses the anti-oxidant effects besides having protective activities in rats.

Key words: , antioxidant enzymes, glutathione S-transferases, cytochrome P450, chemoprotection.

INTRODUCTION

It is generally accepted that reactive oxygen species noids, lycopenes and flavonoids that prevent free radical (ROS) and/or free radicals play an important role in the damages (Stahelin et al., 1991; Steinberg, 1991; Willett, development of tissue damage and pathological events in 1994). Thus, much attention has been focused on the living organisms (Kehrer, 1993; Halliwel and Gutteridge, investigation of antioxidants that can scavenge ROS, 1999). There are increasing evidences that increased especially natural antioxidants, phenolic and flavonoids consumption of and vegetables and intake of from which are mostly used as protective agents certain nonnutrients that are present in foods reduce the against free radical-mediated diseases (Shahidi and risk of various pathological events such as cancer Wanasundra, 1992; Rice-Evans et al., 1996). In addition, (Goodwin and Brodwick, 1995; Steinmetz and Potter, the traditional use of medicinal plants or their crude 1996) and cardio- and cerebro-vascular diseases (Rimm extracts in the prevention and/or treatment of several et al., 1996). This is often attributed to the antioxidants in chronic diseases has been increasingly practiced in the fruits and vegetables such as C, E, carote various different ethnic societies worldwide. This study was designed to specifically investigate the antioxidant and chemoprotective efficacy of Momordica charantia fruit extract by investigating antioxidant *Corresponding author. E-mail: [email protected] Tel: enzymes, cytochrome P450s (CYPs) and glutathione S- +90-258-213-4030/1456. Fax: +90-258-212-5546 transferases (GSTs) in Wistar rat liver, lung and kidney 274 Afr. J. Biotechnol.

tissues when it is administered in vivo. near the Edremit Bay between Ayvalik and Edremit. The antioxidant enzymes (AOE) include superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase Extraction (GPx) and indirectly glutathione reductase (GR). Their role as protective enzymes are well known and have The collected fruits, air-dried and fine powdered, were extracted been extensively investigated both in vivo and in vitro in with hexane using soxhlet extraction apparatus according to the model systems. Cytochrome P450 (CYP, EC 1.14.14.1) Soxhlet’s method where materials are extracted by repeated enzymes are active in monooxygenation and percolation which lasts about 6 - 8 h with hexane under reflux in a special glassware. At the end of extraction process, the solvent is hydroxylation of various xenobiotics, including drugs, then evaporated and the remaining mass is measured. The carcinogens, environmental pollutants as well as many percentage yields are calculated as mg per g dried fruits. Finally, endogenous substrates, fatty acids, arachidonic acids, residues were dissolved in dimethysulfoxide (DMSO) : water (8:2) steroids, prostaglandins, leukotrienes (Nebert and mixture. Gonzales, 1987; Zimniak and Waxman, 1993). Glutathione S-transferases (GST, EC 2.5.1.18) are a Animal complex multigene family of enzymes that are widely distributed in the animal kingdom (Snyder and Maddison, Male Wistar rats (12-14 weeks old) weighing 200-250 g were used. 1997). The most important function of GST is They were housed University Animal House in standard conditions detoxification, conjugating reduced glutathione with a and fed with standard diet with water ad libitum. All experimental large number of electrophilic metabolites derived from a procedures in animals such as administration of substances by i.p., variety of xenobiotics, including carcinogens, toxins and collection of blood and tissue etc. are performed to the national standards under appropriate regimes with Veterinary services and drugs. GSTs are also involved in the metabolism of licensed projects. Rats were randomly assorted into the following endogenous substances such as leukotriene and four groups (each group consisting of 8-20 rats). Group I (control) prostaglandins. some rats were treated with DMSO-water mixture, i.p. daily for 4 Momordica charantia L. () known as days; Group II (Mom) rats were treated with M. charantia fruit bitter melon, balsam pear, Karela, etc. is a vegetable extract 200 mg/kg, i.p. daily for 4 consecutive days; Group III (CCl4) rats were administered with CCl to induce hepatotoxicity at dose of indigenous to tropical areas including India, Asia, South 4 10 mg CCl4/kg, i.p. daily for 2 consecutive days; and Group IV America and and cultivated throughout South (Mom + CCl4) rats were pretreated with 200 mg/kg; i.p. daily for 4 America as food and medicine. Various preparations of consecutive days prior to administration of CCl4 10 mg CCl4/kg, i.p. M. charantia from injectable extracts to fruit juice to dried daily for 2 consecutive days. fruit bits have been traditionally used worldwide, At the end of experimental period, the rats were anesthetized particularly for blood- lowering effects (Welihinda et with ether following a 16-hr fasting. Blood samples were taken from aorta to determine the serum enzymes. The livers, lungs and al., 1986; Raman and Lau, 1996). In addition, it has been kidneys were removed and rinsed with cold physiological saline and reported to exhibit diverse biological activities such as stored at -80°C until analyzed. antioxidant, antimicrobial, antiviral, antihepatotoxic and antiulcerogenic activities which are attributed to an array of biologically active plant chemicals including Chemicals triterpenes, pisteins and steroids (Grover and Yadav, 1-chloro-2,4-dinitrobenzene (CDNB), alanine, aspartic acid, bovine 2004). serum albumin (BSA), glutathione reduced (GSH), H2O2, Although M. charantia is not a native plant in Turkey, glutathione reductase, methoxy- and ethoxyresorufin, cholic acid, - the fruits, known as “kudret nari”, are frequently used in nicotinamide adenine dinucleotide phosphate (-NADPH) and folk medicine, especially in west and southwest part of pyrogallol were purchased from Sigma (USA). All other chemicals Anatolia for the treatment of peptic ulcers and tumors. and solvents were of highest purity and analytical grade.

Recently, the plant is being cultivated in Western Anatolia. In the view of ethnomedical reports M. charantia Preparation of tissues subcellular fractions is being used in folkloric medicine on various ulcers, and infections (Gurbuz et al., 2000; Scartezzini The tissues were homogenized in 5 parts homogenization solution and Speroni, 2000; Beloin et al., 2005). Thus, present (1.15% KCl containing 250 mM EDTA, 100 mM phenylmethylsulphonylfluoride, 100 mM butylated hydroxytoluene, study was undertaken to further characterize the 0.025% cholate) using a tissue homogenizer with a teflon pestle at biological activities of M. charantia. 4°C. The subcellular fractions of rat tissues were prepared by a standard differential centrifugation procedure as described previously (Sen and Kirikbakan, 2004). The amounts of proteins in MATERIALS AND METHODS various fractions were measured using the method of Lowry et al. (1951) with BSA as the standard.

Plant material Determination of total antioxidant response The fruits of M. charantia were collected in the fall of 2003 and 2004 from the foot of Kazdaglari (Mount Ida), Balikesir, Turkey. Antioxidant capacity of M. charantia was determined by measuring Mount Ida, which is also very popular in mythology due to its role total antioxidant response (TAR) against potent free radical reaction in legendary Trojan War, is 1774 m. Height Mountain and is located as described by Erel (2004). Semiz and Sen 275

Determination of serum AST and ALT and LDH activities liver of rats challenged with CCl4 as compared to control. Therefore, these results demonstrated that the fruit of M. In order to evaluate hepatocellular damage and antihepatotoxic charantia has protective function against CCl toxicity in potential of the M. charantia fruit extract enzymatic leakage of 4 transaminases [ALanine aminoTransferase (ALT) and ASpartate rat liver when administered in vivo at a dose of 200 mg / aminoTransferase (AST)] and Lactate DeHydrogenase (LDH) in kg/day i.p, for 4 consecutive days prior to CCl4 intoxica- serum were measured by the method of Raitman and Frankel tion. Similar results have been reported for some other (1957) and Wroblewski and La Due (1955), respectively. Blood was ethnobotanical fruits and herbs (Hsiao et al., 2003; Kim et centrifuged at 4000 rpm at 4°C for 10 min to get serum. al., 2003; Jung et al., 2004).

Antioxidant actions of food may be through inhibitory Determination of antioxidant enzyme activities actions on generation of ROS or by direct scavenging of free radicals. In addition, the levels of endogenous antio- SOD activity was measured by the method of Marklund and xidants may also be up regulated by increasing expres- Marklund (1974). CAT activity was measured using Abei’s method (1974). The GPx activity was assayed by using Paglia and sion of the genes encoding the antioxidant enzymes Valentine’s method (1967). SOD, CAT and GPx. AOE and antioxidant molecules can inhibit free radical production by chelating the transition

Determination of phase I and phase II enzyme activities metal catalysts, breaking chain reactions, reducing concentrations of ROS and/or scavenging initiating radi- Cytochrome P450 dependent 7-ethoxyresorufin O-deethylase cals (Aruoma, 1994; Halliwel and Gutteridge, 1999). In (EROD) and 7-methoxyresorufin O-deethylase (MROD) activities order to investigate whether the antioxidant activities of were determined by the fluorometric measurements of resorufin M. charantia fruit and its related constituents are media- formed using the method of Burke and Mayer (1974) as optimized ted by an increase in antioxidant enzymes, we measured by Sen and Arinc (2000). GST activities using CDNB as substrate were determined as described by Habig et al. (1974). SOD, CAT and GPx and GST activities in different tissues of rats treated with M. charantia fruit extract. In the present study, treatment of rats with M. charantia fruit Statistical analysis extract significantly increased rat liver, lung and kidney SOD, CAT and GPx activities (Table 2). There were Results are expressed as mean ± SD of at least three determinations for each data point. One way ANOVA, Tukey and significant (p<0.001) increase in all hepatic CAT, SOD Dunnett tests were applied for analyzing the significance of differ- and GPx activities. More than four-fold increases were ence between and among different groups. observed with M. charantia fruit extract treatment in hepatic antioxidant enzymes suggesting that liver plays predominant role in protection against free radicals. RESULTS AND DISCUSSION Although similar significant levels of elevations were

observed in GPx activities in different tissues, CAT and M. charantia fruit extract was studied for its antioxidant SOD activities were found to be increased to lesser and chemoprotective effects by investigating antioxidant degrees in kidney and lung tissues (Table 2). SOD, CAT enzymes, cytochrome P450s and glutathione S-transfe- and GPx are major free radical scavenging enzymes that rases as well as its protective effects on hepatocellular have shown to be reduced in a number of pathophy- damage in CCl -intoxicated rats were evaluated. 4 siological processes and diseases such as diabetes Free radical scavenging effects of M. charantia fruit (Cohen and Heikkila, 1994). Thus, activation of these extract was tested by measuring total antioxidant status AOE by the administration of M. charantia fruit extract of M. charantia using a colorimetric method for the total clearly shows that M. charantia fruit contains free radical antioxidant response (TAR) as described by Erel (2004). scavenging activity, which could exert a beneficial action The relative total antioxidant response of M. charantia against pathophysiological alterations caused by the was determined 1.43 ± 0.02 mmol Trolox equiv/L, which presence of superoxide and hydroxide radicals. was higher than 1 mM GSH, indicating potential antioxi- In addition M. charantia fruit extract caused significant dant properties of M. charantia fruit extract. The effects of (p<0.01) elevations on hepatic and nephritic cytosolic M. charantia extract on the serum transaminases and GSTs which are responsible for the metabolism of LDH in control and CCl -intoxicated rats are given in 4 numerous xenobiotics and play a major cellular antioxi- Table 1. Treatment of rats with M. charantia fruit extract dant role. GSTs also have peroxidase and isomerase alone did not alter the enzyme activities while in the CCl - 4 activity; they bind covalently with reactive metabolites intoxicated groups transaminases (ALT and AST) and formed from carcinogens and noncovalently with LDH activities significantly increased (p<0.001). However, lipophilic compounds, thereby offering protection against the rats pre-treated with M. charantia decreased signifi- oxidative stress (Hayes and Pulford, 1995). cantly these elevated transaminases and LDH activities In this study we have also attempted to examine (Table 1). CCl is a known hepatotoxic compound work- 4 whether M. charantia fruit extract were able to affect ing through the generation of reactive free radicals. some CYP isoforms in male rat livers by using highly Treatment with M. charantia fruit extract was found to up specific substrates to CYP isozymes. Treatments of rats regulate different antioxidant and detoxifying enzymes in 276 Afr. J. Biotechnol.

Table 1. The effects of M. charantia fruit extracts on serum LDH, AST and ALT.

Enzyme activities (Units) Groups LDH AST ALT Control 670 ± 96 188.85 ±29 152.22 ± 19 Momordica 690 ± 90* 142.93 ± 15* 117.78 ± 14*

CCl4 1,680 ± 166*** 528.30 ± 74*** 406.81 ± 63***

CCl4 + Momordica 210 ± 15* 185.33 ± 21* 164.17 ± 16*

* p<0.05, ** p<0.01, *** p<0.001.

Table 2. The effects of M. charantia fruit extracts on anti-oxidant enzyme activities.

Liver Kidney Lung Enzymes Control Momordica Control Momordica Control Momordica CAT (nmol/min/mg ) 19.47 ± 2.79 92.01 ± 9.13*** 11.79 ± 2.79 24.17 ± 3.17** 14.24 ± 1.84 3.12 ± 0.87* GPx (nmol/min/mg protein) 0.96 ± 0.26 4.77 ± 0.44*** 0.41 ± 0.10 3.42 ± 0.13*** 1.18 ± 0.24 2.67 ± 0.19*** GST (nmol/min/mg protein) 257.8 ± 13.6 365.9 ± 27.6** 77.6 ± 4.4 150.3 ± 7.5** 156.7 ± 6.8 148.2 ± 10.9* SOD (Unit/mg protein) 1.21 ± 0.06 5.09 ± 0.09*** 0.23 ± 0.05 0.28 ± 0.05** ND ND

* p<0.05, ** p<0.01, *** p<0.001.

with M. charantia significantly reduced both EROD Table 3. The effects of M. charantia fruit extracts on CYP1A (p<0.01) and MROD (p<0.05) activities in rat liver micro- dependent enzyme activities. somes, which are known to be catalyzed by CYP1A iso- forms (Table 3). Although species differences make it Enzyme activities (pmol resorufin/min/mg difficult to extrapolate the inhibitions from rats to human, Groups protein) selective inhibition of CYP1A isoforms could be consi- EROD MROD dered as one possible mechanism of the chemo-protec- Control 106.5 ± 9.62 47.07 ± 3.53 tive action of M. charantia because they are pri-marily Momordica 70.51 ± 9.57** 41.59 ± 8.16* involved in the metabolism and activation of carci-nogens and procarcinogens (Ioannides and Lewis, 2004). In * p<0.05, ** p<0.01, *** p<0.001 addition, caution should be paid to the possible drug interactions in people who concurrently using M. charan- tia. However, further studies are required to be carried ACKNOWLEDGEMENTS out on this CYP field to clearly elucidate which isoforms and how they are involved in the modulatory eff-ects. This work is supported by the Scientific Research These findings clearly demonstrated the antioxidant Projects Council of Pamukkale University (PAUBAP- and chemoprotective activities of M. charantia fruit extract FEF2003-014). We would also like to thank Turkish Aca- in experimental rat models. Although further studies are demy of Sciences. required to identify active components involved in the antioxidant and chemoprotective activity of this fruit, results strongly suggest that M. charantia fruit extract has REFERENCES chemoprotective action against CCl -induced toxicity. In 4 Abei H (1974). Catalase. In Method of Enzymatic Analysis. New York: addition, even if it is indirect, inhibition of CYP1A Academic Press, pp: 673-684. dependent activities could be considered as a promising Aruoma OI (1994). Nutrition and health aspects of free radicals and cancer chemoproventive action by lowering metabolic antioxidants. Food Chem. Toxicol. 32: 671-683. activation of various carcinogens and/or procarcinogens. Beloin N, Gbeassor M, Akpagana K, Hudson J, de Soussa K, Koumaglo K, Arnason JT (2005). Ethnomedicinal uses of Momordica charantia Based on our knowledge, the present study is the first (Cucurbitaceae) in Togo and relation to its phytochemistry and reporting the effect of M. charantia fruit extract on CYP1A biological activity. J. Ethnopharmacol. 96: 49-55. dependent enzyme activities, namely EROD and MROD. Burke MD, Mayer RT (1974). Ethoxyresorufin: Direct fluorometric assay In conclusion, the present study showed that in vivo of microsomal O-dealkylation which is preferentially induced by 3- methylcholanthrene. Drug Metab. Dispos. 2: 583-588. treatment of rats with M. charantia fruit extract enhanced Cohen G, Heikkila RE (1974). The generation of hydrogen peroxide, both AOE and GST activities while lowering CYP1A superoxide radical, and hydroxyl radical by 6-hydroxydopamine, dependent enzyme activities. dialuric acid, and related cytotoxic agents. J. Biol. Chem. 249: 2447- 2452. Semiz and Sen 277

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